J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHalpha) couplings in 15N-labeled proteins

A new method for the measurement of homonuclear 3J(HNHalpha) coupling constants in 15N-labeled small proteins is described. The method is based on a modified sensitivity enhanced HSQC experiment, where the 3J(HNHalpha) couplings are multiplied in the f1-dimension. The J-multiplication of homonuclear 3J(HNHalpha) couplings is based on simultaneous incrementation of 15N chemical shift and homonuclear coupling evolution periods. The time increment for the homonuclear coupling evolution period is chosen to be a suitable multiple (2N x t1) of the corresponding increment for 15N-shift evolution. This results in the splitting of the HSQC correlation in the f1-dimension by 2N x 3J(HNHalpha). Because the pulse sequence has good sensitivity and water suppression properties, it is particularly useful for natural abundance samples.     

Accurate measurement of H(N)-H(alpha) residual dipolar couplings in proteins.

A method for accurately measuring H(N)-H(alpha) residual dipolar couplings is described. Using this technique, both the sign and magnitude of the coupling can be determined easily. Residual dipolar coupling between H(N)(i)-H(alpha)(i) and H(N)(i)-H(alpha)(i-1) were measured for the FK506 binding protein complexed to FK506. The experimental values were in excellent agreement with predictions based on an X-ray crystal structure of the protein/ligand complex, suggesting that these residual dipolar couplings will provide accurate structural constraints for the refinement of protein structures determined by NMR.     

Cosine modulated HSQC: a rapid determination of (3)J(HNHalpha) scalar couplings in (15)N-labeled proteins

A two-dimensional HSQC-based NMR method, (15)N-COSMO-HSQC, is presented for the rapid determination of homonuclear (3)J(HNHalpha) couplings in (15)N-labeled proteins in solution. Scalar couplings are extracted by comparing the intensity of two separate datasets recorded with and without decoupling of the (3)J(HNHalpha) during a preparation period. The scalar couplings are introduced through a cosine modulation of the peak intensities. The experiment relies on a BIRD sandwich to selectively invert all amide protons H(N) and is very simple to implement. (3)J(HNHalpha) couplings were determined using both the (15)N-COSMO-HSQC and quantitative-J on (15)N-labeled chemokine RANTES. The two experiments show well-correlated values.     

Improved measurement of (3)J(H(alpha)(i),N(i+1)) coupling constants in H(2)O dissolved proteins.

A modification to the recently proposed alpha/beta-HN(CO)CA-J TROSY pulse sequence (P. Permi et al., J. Magn. Reson. 146, 255-259 (2000)) makes it possible to determine (3)J(H(alpha)(i), N(i+1)) coupling constants from a single E.COSY-type cross-peak pattern rather than from two (1)H(alpha) spin-state-edited subspectra. Advantages are increased (15)N resolution, critical to extracting accurate (1)H(alpha)-(15)N coupling constants, and minimized differential relaxation due to nested (13)C(alpha) and (15)N evolution periods. Application of the improved pulse sequence to Desulfovibrio vulgaris flavodoxin results in (3)J(H(alpha)(i), N(i+1)) values being systematically larger than those obtained with the original scheme. Parametrization of the coupling dependence on the protein backbone torsion angle psi yields the Karplus relation (3)J(H(alpha)(i), N(i+1))=-1.00 cos(2)(psi-120 degrees )+0.65 cos(psi-120 degrees )-0.15 Hz, with a residual root-mean-square difference of 0.13 Hz between measured and back-calculated coupling constants. The curve compares with data derived from ubiquitin (A. C. Wang and A. Bax, J. Am. Chem. Soc. 117, 1810-1813 (1995)), although spanning a slightly larger range of J values in flavodoxin. The orientation of the Ala39/Ser40 peptide link, forming a type-II beta-turn in flavodoxin, is twisted against X-ray-derived torsions by approximately 10 degrees in the NMR structure as evident from the analysis of straight phi- and psi-related (3)J coupling constants. The remaining deviation of some experimental values from the prediction is likely to be due to strong hydrogen bonding, substituent effects, or the additional dependence on the adjacent torsions straight phi.     

Efficient measurement of (3)J(N,Cgamma) and (3)J(C',Cgamma) coupling constants of aromatic residues in (13)C, (15)N-labeled proteins

An NMR pulse sequence is proposed for the simultaneous determination of side chain chi1 torsion-angle related (3)J(N,Cgamma) and (3)J(C', Cgamma) couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated (15)N and (1)H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly (13)C/(15)N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.     

Measurement of (1)J(NC') and (2)J(H(N))(C') couplings from spin-state-selective two-dimensional correlation spectrum.

A method for the measurement of (1)J(NC') and (2)J(H(N))(C') coupling constants from a simplified two-dimensional [(15)N, (1)H] correlation spectrum is presented. The multiplet components of the (1)J(NC') doublet in the indirect dimension and (2)J(H(N))(C') in the direct dimension are separated into two subspectra by spin-state-selective filters. Thus each subspectrum contains no more peaks than the conventional [(15)N, (1)H]-HSQC spectrum. Furthermore, the method for the measurement of (1)J(NC') and (2)J(H(N))(C') is designed to exploit destructive relaxation interference (TROSY). The results are verified against the measurements of (1)J(NC') from spin-state-selective [(13)C', (1)H] correlation spectra recorded with additional sequence described here.     

Semi-Constant-Time HMSQC (SCT-HMSQC-HA) for the Measurement of JHH Couplings in N-Labeled Proteins

A simple method for accurately measuring ³J_H^NH^α coupling constants in ¹⁵N-labeled proteins is described. This semi-constant-time HMSQC-HA experiment combines the rapidity and convenience of the recently introduced CT-HMQC-HA scheme (Postingl and Otting, J. Biomol. NMR 12, 319–324 (1998)) with the high resolution and robustness of the HSQC experiment. The proposed method is demonstrated for the 76-residue human ubiquitin and Saccharopolyspora erythraea calerythrin (176 residues). Our results imply that the SCT-HMSQC-HA experiment is suitable also for proteins with less favorable NMR properties due to its good resolution and sensitivity.     

Determination of internucleotide (h)J(HN) couplings by the modified 2D J(NN)-correlated [(15)N, (1)H] TROSY

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J(NN)-correlated [(15)N, (1)H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147-14151, 1998) can be applied to measure (h)J(HN) in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA(Trp), since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J(NN)-correlated [(15)N, (1)H] TROSY experiment which enables direct measurement of (h)J(HN) in large nucleic acids.     

Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N-1H(N) and 15N-13C' and two-bond 13C'-1H(N) residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N-1H(N) and 15N-13C(') and two-bond 13C(')-1H(N) residual dipolar couplings in proteins. Compared with existing experiments, the (delta,J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.     

3hJ coupling between C(alpha) and H(N) across hydrogen bonds in proteins

J couplings between (13)C(alpha) and (1)H(N) across hydrogen bonds in proteins are reported for the first time, and a two- or three-dimensional NMR technique for their measurement is presented. The technique exploits the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms, for sensitivity enhancement. The 2D or 3D spectra exhibit E.COSY patterns where the splittings in the (13)CO and (1)H(N) dimensions are (1)J((13)C(alpha), (13)CO) and the desired (3h)J((13)C(alpha), (1)H(N)), respectively. A demonstration of the new method is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2 where 17 (3h)J((13)C(alpha), (1)H(N)) coupling constants ranging from 0 to 1.4 Hz where identified and all of positive sign.     

Methods for the measurement of (1)J(NCalpha) and (2)J(NCalpha) from a simplified 2D (13)C(alpha)-coupled (15)N SE-HSQC spectrum

Two methods for the measurement of (2)J(NCalpha) and (1)J(NCalpha) in (15)N/(13)C-labeled small and medium-size proteins are described. The current approach is based on simplified (13)C(alpha)-coupled (15)N HSQC spectra, where the two (2)J(NCalpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NCalpha) doublets between the two subspectra provides an accurate value for (1)J(NCalpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation.     

New (15)N NMR exchange experiments for the unambiguous assignment of (1)H(N)/(15)N resonances of proteins in complexes in slow chemical exchange with free form

The potentialities of a 2D proton-detected heteronuclear exchange experiment to assign the nitrogen and amide proton resonances in a uniformly (15)N-enriched macromolecule involved in a complex, starting from the free form assignments, are demonstrated on a protein-DNA complex. This 2D experiment is further extended to a 3D experiment in the case of severe superpositions.     

Suppression of diagonal peaks in TROSY-type 1H NMR NOESY spectra of 15N-labeled proteins.

A novel method for suppression of diagonal peaks in the amide region of NOESY NMR spectra of 15N-labeled proteins is presented. The method is particularly useful for larger proteins at high magnetic fields where interference between dipolar and chemical shift anisotropy relaxation mechanisms results in large TROSY effects, i.e. , large differences in 1HN linewidths depending on the spin state of attached 15N nuclei. In this limit the new TROSY NOESY method does not compromise sensitivity. It is demonstrated using a perdeuterated 15N-labeled protein sample, Neural Cell Adhesion Molecule 213-308 (NCAM) from rat, in H2O at 800 MHz.     

New techniques for the measurement of C'N and C'H(N) J coupling constants across hydrogen bonds in proteins

Two new two- or three-dimensional NMR methods for measuring (3h)J(C'N) and (2h)J(C'H) coupling constants across hydrogen bonds in proteins are presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D or 3D spectra into two separate subspectra corresponding to the two possible spin states of the (1)H(N) spin during evolution of (13)CO coherences. This allows (2h)J(C'H) to be measured in an E.COSY-type way while (3h)J(C'N) can be measured in the so-called quantitative way provided a reference spectrum is also recorded. A demonstration of the new methods is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2.     

Measurement of relaxation rates of N(H) and H(alpha) backbone protons in proteins with tailored initial conditions.

Several methods are presented for the selective determination of spin-lattice and spin-spin relaxation rates of backbone protons in labeled proteins. The relaxation rates of amide protons in (15)N labeled proteins can be measured by using two-way selective cross-polarization (SCP). The measurement of H(alpha) relaxation rates can be achieved by combining this method with homonuclear Hartmann-Hahn transfer using doubly selective irradiation. Various schemes for selective or nonselective inversion of the longitudinal proton magnetization lead to different initial recovery rates. The methods have been applied to lysine K6 in (15)N-labeled human ubiquitin and to leucine L5 in (15)N- and (13)C-labeled octapeptide YG*G*F*LRRI (GFL) in which the marked residues are (15)N- and (13)C-labeled.     

Improved measurement of (15)N-[(1)H] NOEs in the presence of H(N)-water proton chemical exchange.

A simple method is presented to accurately determine (15)N-[(1)H] NOEs in biomolecules in the presence of H(N)-water proton chemical exchange. Three measurements are required: one with nonselective proton saturation and two with different water saturation conditions to determine the equilibrium value of the (15)N signal. This approach is exemplified with data on two peptides, one helix-forming 17-mer and one compactly folded 56-mer. Results indicate that (15)N-[(1)H] NOEs determined using the standard approach with short recycle times (3 to 4 s) can be significantly in error when H(N)-water proton chemical exchange is relatively rapid, water proton relaxation is relatively slow, and (15)N-[(1)H] NOEs are away from the value of -1. This new method avoids such inaccuracies resulting from the use of short recycle times.     

The role of (15)N CSA and CSA/dipole cross-correlation in (15)N relaxation in solid proteins

The influence of the (15)N CSA on (15)N longitudinal relaxation is investigated for an amide group in solid proteins in powder form under MAS. This contribution is determined to be typically 20-33% of the overall longitudinal relaxation rate, at 11.74 and 16.45 T, respectively. The improved treatment is used to analyze the internal dynamics in the protein Crh, in the frame of a motional model of diffusion in a cone, using the explicit average sum approach. Significant variations with respect to the determined dynamics parameters are observed when properly accounting for the contribution of (15)N CSA fluctuations. In general, the fit of experimental data including CSA led to the determination of diffusion times (tau(w)) which are longer than when considering only an (15)N-(1)H dipolar relaxation mechanism. CSA-Dipole cross-correlation is shown to play little or no role in protonated solids, in direct contrast to the liquid state case.     

Triple-resonance experiments for correlation of H5 and exchangeable pyrimidine base hydrogens in (13)C,(15)N-labeled RNA.

Triple-resonance two-dimensional H5(C5C4N)H experiments are described that provide through-bond H5 to imino/amino connectivities in uridines and cytidines in (13)C, (15)N-labeled RNAs. The experiments employ selective INEPT steps for transferring magnetization from the H5 hydrogens through the intervening C5, C4, and N3/N4 nuclei to the imino/amino hydrogens. The improved sensitivity of these experiments for assignments in a large 43-nucleotide RNA is demonstrated.     

On the accurate measurement of amide one-bond 15N-1H couplings in proteins: effects of cross-correlated relaxation, selective pulses and dynamic frequency shifts

Amide one-bond 15N-1H scalar couplings of 15N- and [15N,2H]-isotopically enriched ubiquitin have been measured with the Quantitative J approach by monitoring NMR signal intensity modulation. Scalar couplings of the non-deuterated protein are in average approximately 0.6 Hz larger than values of deuterated ubiquitin. This deviation is 30 times the error derived from experiment reproducibility. Refocusing dipole/dipole cross-correlated relaxation decreases the discrepancy to approximately 0.1 Hz, suggesting that it likely originates from relaxation interference. Alternatively, the subtraction of J values obtained at different magnetic fields largely reduces the relaxation effects. In contrast, the dynamic frequency shift whose main contribution to 1J(15N-1H) arises from 15N chemical shielding anisotropy/NH dipole cross-correlation, is not eliminated by refocusing spin evolution under this interaction. Furthermore, the average difference of 1J(15N-1H) values at two magnetic fields closely agrees with the theoretical expected difference in the dynamic frequency shift.     

Isolation of NO(3)(-)-N as 1-phenylazo-2-naphthol (Sudan-1) for measurement of delta(15)N

The conversion of nitrate (NO(3)(-)) to 1-phenylazo-2-naphthol (Sudan-1) has been examined as a method for natural abundance measurement of delta(15)N of NO(3)(-). The reaction results in dilution of NO(3)(-)-N with only one reagent-derived N and the product is readily concentrated from dilute samples by reverse phase chromatography. There is systematic isotopic fractionation during the reaction, but this can be allowed for by analysing known NO(3)(-) standards along with each sample set. Sudan-1 prepared from surface water samples containing approximately 50 &mgr;g NO(3)(-)-N can be analysed by automated continuous flow isotope ratio mass spectrometry with a precision of 0.2 per thousand (one standard deviation) and the accuracy is not affected by interference from other nitrogenous species in the sample or reagents. Copyright 1999 John Wiley & Sons, Ltd.